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Background: miRNAs have enormous potential to be used as diagnostic and prognostic markers as well as therapeutic targets in male infertility and diseases of the reproductive system. This study aimed to investigate the association between the two functional genetic variants in the hsa-miR27a (rs2910164) and hsa-miR-146a gene (rs895819) and male infertility in North Macedonian population, as well as to test their association with the values of major seminal parameters. Methods: The case group included in this study comprised 158 men initially diagnosed with idiopathic male infertility. The control group included 126 age-matched healthy male volunteers who fathered at least one child. Results: We report the association of rs2910164 minor allele C for the first time with the increased susceptibility to asthenoteratozoospermia. Additionally, our results indicating the association of allele C with low sperm vitality are a novel finding. We did not demonstrate the association between genetic variant rs895819 and the risk of different types of male infertility. Still, the number of participants with CC genotype in subjects diagnosed with asthenoteratozoospermia was null, while in controls, it reached 7.2%. We further detected the rs895819 genotype-dependent difference in rapid progressive sperm motility. Conclusions: The association of rs2910164 and rs895819 with idiopathic male infertility in general is unlikely. However, both of these variants show an association with certain types of male infertility and with sperm abnormalities, which need to be confirmed in later studies in different ethnic groups.

After sample processing for RNA and DNA analysis, the leftover protein pellets are usually discarded due to the limited efficiency of pellet reconstitution/solubilisation. As the pelleted proteins are tightly packed, they are most often solubilised using chaotropic agents (e.g., guanidine hydrochloride or urea), detergents (e.g., SDS), salts (NaCl) or basic buffer (Tris). The aim of this study was to define and optimise the procedure for the efficient extraction of proteins from human peripheral blood mononuclear cells (PBMCs), obtained by a single blood draw and lysed in TRIzol reagent, by varying experimental conditions in terms of protein precipitation solvent (isopropanol or acetone), washing (with or without guanidine hydrochloride) and solubilisation solution (containing SDS, NaCl, urea and/or Tris). We evaluated the efficacy of the final, optimised protocol to solubilise both small cytoplasmic and larger transmembrane proteins, and the compatibility with methods employed for the subsequent analysis of protein posttranslational modifications, such as glycosylation. The optimised protocol for the extraction and isolation of post-TRIzol leftover proteins from PBMCs can be defined as follows: protein precipitation from the organic phase with ice-cold acetone, pellet washing with absolute ethanol and solubilisation in 1 % SDS, employing 20 min heating at 50ºC and vortexing.

Recent findings highlighted the potential of long non-coding RNAs (lncRNAs) as novel indicators of gestational diabetes mellitus (GDM), as they demonstrate altered expression in metabolic disorders, oxidative stress (OS) and inflammation (IFM). The aim of this study was to evaluate the diagnostic potential and prognostic significance of the OS/IFM-related lncRNAs H19, MALAT1 and MEG3 in GDM and their correlations with redox status-related parameters. The relative quantification of selected lncRNAs from peripheral blood mononuclear cells (PBMCs) of GDM patients and controls (n = 50 each) was performed by qPCR. The expression levels were tested for correlations with metal ion concentrations, NRF2 expression, activities of glutathione reductase (GR), superoxide dismutase (SOD), catalase (CAT), serum thiol content, protein carbonyl level and thiobarbituric acid reactive substances. MALAT1 and H19 were significantly downregulated in GDM patients ( p = 0.0095 and p = 0.012, respectively). A correlation was observed between H19 expression and zinc levels in both GDM patients and controls. MALAT1 expression positively correlated with NFE2L2 levels in GDM patients ( p = 0.026), while H19 exhibited a positive correlation with GR activity in controls ( p = 0.018) and an inverse correlation with SOD activity ( p = 0.048). Our data show the disturbance of OS/IFM-lncRNAs in GDM pathogenesis and illustrate the biomarker potential of the analyzed lncRNAs, as well as of certain redox status parameters.

Background and Objectives : Gestational diabetes mellitus (GDM) may impact both maternal and fetal/neonatal health. The identification of prognostic indicators for GDM may improve risk assessment and selection of patient for intensive monitoring. The aim of this study was to find potential predictors of adverse pregnancy outcome in GDM and normoglycemic patients by comparing the levels of different biochemical parameters and the values of blood cell count (BCC) between GDM and normoglycemic patients and between patients with adverse and good outcome. Materials and Methods : Prospective clinical study included 49 patients with GDM (study group) and 44 healthy pregnant women (control group) who underwent oral glucose tolerance test (OGTT) at gestational age of 24-28 weeks. At the time of OGTT peripheral blood was taken for the determination of glucose levels, insulin, glycated hemoglobin, lipid status, homeostatic model assessment, BCC, iron and zinc metabolism, liver function, kidney function and inflammatory status. Each group was divided into two subgroups-normal and poor pregnancy outcome. Results : Higher RBC, hemoglobin concentration, hematocrit value, fasting glucose, uric acid and fibrinogen were found in GDM patients compared to control group. In GDM patients with poor pregnancy outcome values of fibrinogen, ALT, sedimentation rate, granulocyte and total leukocyte counts were elevated, while the serum level of zinc was significantly lower. Higher level of fibrinogen was found in normoglycemic patients with adverse pregnancy outcomes. ROC curve was constructed in order to assess fibrinogen's biomarker potential. The established AUC value for diagnostic ROC was 0.816 ( p < 0.001, 95% CI 0.691-0.941), while the AUC value for assessing fibrinogen's potential to predict poor pregnancy outcome in GDM was 0.751 ( p = 0.0096, 95% CI 0.561-0.941). Conclusions : The results of our study demonstrated that the best prognostic potential in GDM showed inflammation related parameters, identifying fibrinogen as a parameter with both diagnostic and prognostic ability.

Chronic kidney disease (CKD) is a global health concern affecting approximately one billion individuals worldwide. End-stage kidney disease (ESKD), the most severe form of CKD, is often accompanied by anemia. Peritoneal dialysis (PD), a common treatment for ESKD, utilizes the peritoneum for solute transfer but is associated with complications including protein loss, including transferrin (Tf) a key protein involved in iron transport. This study investigated Tf characteristics in ESKD patients compared to healthy individuals using lectin microarray, spectroscopic techniques and immunocytochemical analysis to assess Tf interaction with transferrin receptors (TfRs). ESKD patients exhibited altered Tf glycosylation patterns, evidenced by significant changes in lectin reactivity compared to healthy controls. However, structural analyses revealed no significant differences in the Tf secondary or tertiary structures between the two groups. A functional analysis demonstrated comparable Tf-TfR interaction in both PD and healthy samples. Despite significant alterations in Tf glycosylation, structural integrity and Tf-TfR interaction remained preserved in PD patients. These findings suggest that while glycosylation changes may influence iron metabolism, they do not impair Tf function. The study highlights the importance of a glucose-free dialysis solutions in managing anemia exacerbation in PD patients with poorly controlled anemia, potentially offering a targeted therapeutic approach to improve patient outcomes.

Prostate-specific membrane antigen (PSMA) and caveolin-1 are membrane proteins that are overexpressed in prostate cancer (PCa) and are involved in tumor growth and increase in aggressiveness. The aim of the present study is therefore to evaluate PSMA and caveolin-1 proteins from plasma exosomes as effective liquid biopsy biomarkers for PCa. This study included 39 patients with PCa and 33 with benign prostatic hyperplasia (BPH). The shape and size of the exosomes were confirmed by transmission electron microscopy (TEM) and scanning electron microscopy (SEM) analysis. Immunogold analysis showed that PSMA is localized to the membrane of exosomes isolated from the plasma of both groups of participants. The relative protein levels of PSMA and caveolin-1 in the plasma exosomes of PCa and BPH patients were determined by Western blot analysis. The relative level of the analyzed plasma exosomal proteins was compared between PCa and BPH patients and the relevance of the exosomal PSMA and caveoin-1 level to the clinicopathological parameters in PCa was investigated. The analysis performed showed an enrichment of exosomal PSMA in the plasma of PCa patients compared to the exosomes of men with BPH. The level of exosomal caveolin-1 in plasma was significantly higher in PCa patients with high PSA levels, clinical-stage T3 or T4 and in the group of PCa patients with aggressive PCa compared to favorable clinicopathological features or tumor aggressiveness. Plasma exosomes may serve as a suitable object for the identification of potential biomarkers for the early diagnosis and prognosis of PCa as well as carriers of therapeutic agents in precision medicine of PCa treatment.

In previous publications, we pointed out the importance of mannosylation of fibrinogen for the development of cardiovascular complications and fucosylation as a predictor of peritoneal membrane dysfunction in patients on peritoneal dialysis (PD). After a follow-up period of 30 months from the onset of the COVID-19 pandemic, we evaluated the significance of 1,25-dihydroxyvitamin D 3 (calcitriol) therapy, primary disease, biochemical and hematologic analyzes, and previously performed glycan analysis by lectin-based microarray as predictors of mortality in this patient group. After univariate Cox regression analysis, diabetes mellitus (DM) and calcitriol therapy were found to be potential predictors of mortality. Additional multivariate Cox regression analysis confirmed that only DM was a predictor of mortality. Nevertheless, the use of calcitriol in therapy significantly reduced mortality in this patient group, as shown by the Kaplan-Meier survival curve. The presence of DM as a concomitant disease proved to be a strong predictor of fatal outcome in PD patients infected with SARS-CoV-2. This is the first study to indicate the importance and beneficial effect of calcitriol therapy on survival in PD patients with COVID-19 infection. In addition, this study points to the possibility that adverse thrombogenic events observed in PD patients during the pandemic may be caused by aberrant fibrinogen glycosylation.

MiR-21 and miR-375 have been reported as dysregulated in prostate cancer (PCa) in multiple previous studies. Still, variable or even opposing data for the expression of these microRNAs in PCa were found, and their potential biomarker properties remain elusive. In an attempt to clarify their significance as PCa biomarkers, as well as to compare different types of specimens as a source of relevant microRNAs, we used plasma and matching plasma-derived exosomes from patients with PCa and patients with benign prostatic hyperplasia (BPH). Plasma and exosomes were obtained from 34 patients with PCa and 34 patients with BPH, and their levels of expression of miR-21 and miR-375 were determined by RT-qPCR. We found no significant difference in the level of expression of these microRNAs in plasma and exosomes between patients with PCa and BPH. The level of exosomal miR-21 was elevated in PCa patients with high serum PSA values, as well as in patients with aggressive PCa, while for plasma samples, the results remained insignificant. For miR-375, we did not find an association with the values of standard prognostic parameters of PCa, nor with cancer aggressiveness. Therefore, our results support the potential prognostic role of exosomal miR-21 expression levels in PCa.

Insulin-like growth factors (IGFs) are peptides which exert mitogenic, endocrine and cytokine activities. Together with their receptors, binding proteins and associated molecules, they participate in numerous pathophysiological processes, including cancer development. Colorectal cancer (CRC) is a disease with high incidence and mortality rates worldwide, whose etiology usually represents a combination of the environmental and genetic factors. IGFs are most often increased in CRC, enabling excessive autocrine/paracrine stimulation of the cell growth. Overexpression or increased activation/accessibility of IGF receptors is a coinciding step which transmits IGF-related signals. A number of molecules and biochemical mechanisms exert modulatory effects shaping the final outcome of the IGF-stimulated processes, frequently leading to neoplastic transformation in the case of irreparable disbalance. The IGF system and related molecules and pathways which participate in the development of CRC are the focus of this review.

Dysfunctions in mechanisms of gene regulation based on RNA interference are recognized as a common feature of the molecular basis of cancer pathogenesis. Therefore, as one of the crucial components of the machinery involved in the biogenesis of both siRNAs and microRNA molecules, DICER was recognized as one of the candidates for the research in the field of carcinogenesis. Due to their potential functional properties, several genetic variants located within DICER1 gene were analyzed for their possible association with the susceptibility to cancer through case-control studies. In order to elucidate their effect on the overall cancer risk, we conducted an updated meta-analysis of all eligible association studies. The publications were selected based on PubMed database search, while OpenMeta-analyst and MetaGenyo software were used for quantitative data synthesis. Statistically significant results were found for the association of rs1057035 with the overall cancer risk under multiple genetic models (P CT vs. TT  < 0.001, OR CT vs. TT  = 0.870, 95% CI = 0.812-0.933; P allelic  = 0.009, OR allelic  = 0.896, 95% CI = 0.825-0.973; P dom  < 0.001, OR dom  = 0.874, 95% CI = 0.817-0.934; P overdom  = 0.004, OR overdom  = 0.858, 95% CI = 0.773-0.953). Other selected genetic variants within DICER1, rs13078, rs1209904 and rs3742330, did not show the association with the overall susceptibility to malignant diseases. We conclude that rs1057035 may represent a potential biomarker associated with the risk of developing cancer, which requires a confirmation in a larger set of studies., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Glycosylation may strongly affect protein structure and functions. A high risk of cardiovascular complications seen in patients with end-stage renal disease (ESRD) is, at least partly associated with delayed clot formation, increased clot strength, and delayed cloth lysis. Taking into consideration that fibrinogen mediates these processes, we isolated fibrinogen from the plasma from patients with ESRD on peritoneal dialysis (ESRD-PD), and examined glycosylation of native fibrinogen and its subunits by lectin-based microarray and lectin blotting. Compared to healthy controls, fibrinogen from patients had increased levels of A2BG2 and decreased levels of FA2 glycan. The distribution of glycans on individual chains was also affected, with the γ chain, responsible for physiological functions of fibrinogen (such as coagulation and platelet aggregation), being most prone to these alterations. Increased levels of multi-antennary N-glycans in ESRD-PD patients were also associated with the type of dialysis solutions, whereas an increase in the fucosylation levels was strongly related to the peritoneal membrane damage. Consequently, investigation of fibrinogen glycans can offer better insight into fibrinogen-related complications observed in ESRD-PD patients and, additionally, contribute to prognosis, choice of personalised therapy, determination of peritoneal membrane damage, and the length of utilization of peritoneum for dialysis.